Biomass to Hydrogen (B2H2)

Pin-Ching Maness (P.I.)Katherine Chou (Presenter)

National Renewable Energy Laboratory April 30, 2019

DOE Hydrogen and Fuel Cells Program 2019 Annual Merit Review and Peer Evaluation Meeting

P038

This presentation does not contain any proprietary, confidential, or otherwise restricted information.

Overview

Timeline and Budget

• Project start date: 10/1/2015 • FY16 DOE Funding: $1M • FY17 DOE funding: $900K • FY18 DOE funding: $800K • Total DOE funds received to

date: $2.7M • The project will be closed out

in FY19.

Barriers

• H2 molar yield (AX) • Feedstock cost (AY) • System engineering (AZ)

Partners • Dr. Bruce Logan

Pennsylvania State University • Drs. Steven Singer, Lawrence

Berkeley National Lab (LBNL) and Ken Sale, Sandia National Lab (SNL)

• Dr. James Liao at UCLA (no cost)

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NREL

Relevance

Overall Objective: Develop direct fermentation technologies to convert renewable lignocellulosic biomass resources to H2.

Biomass

Dark Fermentation

Acetic, formic, lactic, succinic

acids and ethanol

N1 = 2 – 4 H2

MEC

N2 = 5.8 - 7.6 H2

N1 + N2 = 7.8 – 11.6 mol H2 per mol sugar

Current Project Year Objectives (May 2018 – April 2019) • Addressing feedstock cost barrier

– Improve biomass utilization by converting cellulose (6-carbon sugar) and hemicellulose (5-carbon sugar) to produce H2 either via co-culture systems or genetic engineering of Clostridium thermocellum.

• Addressing H2 molar yield barrier – Had generated a series of competing-pathway mutants with either increased rate or yield

of H2. Using 13C-metabolic flux analysis, we aim to understand how these metabolic changes influence H2 production to guide future genetic engineering strategies.

– Microbial Electrolysis Cells (MEC): Replace the costly platinum cathode with inexpensive materials while still obtaining high rate of H2 production, ultimately using fermentation waste – also addressing waste removal.

This project addresses key DOE Technical Targets and leverages DOE Bioenergy Technologies Office (BETO) investment in biomass pretreatment.

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ApproachTask 1: Bioreactor Performance

• Approach: Optimize bioreactor in batch and fed-batch modes by testing parameters such as corn stover lignocellulose loadings (DMR pretreatment), and hydraulic retention time (HRT), using the cellulose-degrading bacterium Clostridium thermocellum engineered to co-utilize both cellulose and hemicellulose.

Engineer a Cellulose-Degrading Microbe to Co-Metabolize C5 Sugars

Pretreatment – De-acetylated and Mechanically Refined (DMR)

0.4% Solid: 44% cellulose NaOH 23% xylan H2

17% lignin 70°C Ash, etc.

Liquid: 79% acetate 2.9% xylan

C. thermocellum H2 Ferment all the sugars to H2 in one bioreactor NREL | 4

Task 1. Accomplishments and Progress: Increased XylanBiomass Utilization by 29% and H2 Production by 45%.

Optimize and improve biomass utilization by 20% by developing methods to convert FY18 Q4 both C6 and C5 sugars to H2. We will achieve this by ether using C. thermocellum 9/2018 Complete

Milestone already engineered to co-utilize both sugars, or in a binary culture including another microbe to metabolize the C5 sugar of the biomass feedstock

• Use two strategies to improve biomass utilization: − The first approach is to use a binary co-culture system (reported in 2018 AMR). − A second approach is to express foreign xylose-pathways genes (xylAB) to metabolize

xylose, followed by adaptive evolution to utilize xylan (complex xylose). • C. thermocellum evolved xylAB strain (19-9) indeed utilized 29% more xylan and produced

45% more H2 by co-utilizing both cellulose and xylan.

Katherine Chou

Utilizing the hemicellulose portion of biomass will lower feedstock cost. NREL | 5

Accomplishments and Progress : Increased H2 production Rate by 15% and total H2 by 16% in pH-controlled Bioreactor

FY19 Q1 Milestone

Via laboratory evolution, we have evolved the xylose-engineered strain to also degrade the more complex xylan, yet its performance in H2 production has not been demonstrated in bioreactors. We will quantify H2 production in pH-controlled bioreactor and obtain up to 10% increase in H2 production (over a baseline rate of 1 L/L/d at 5 g/L substrate loading) as an indication of xylan utilization. We will also determine xylan utilization and profile metabolites to guide additional engineering strategies to improve xylan utilization.

12/2018

Complete

• The 19-9 evolved strain yielded an average H2 production rate (over 43 h of fermentation) of 1024 mL/L-d using both cellulose and xylan, a 15% increase over the parental controlled strain (∆hpt) using cellulose only, and with a rate of 871 mL/L-d.

• The 19-9 evolved strain also produced 16% more total H2.

Lauren Magnusson

NREL | 6

Accomplishments and Progress : Increased XylanConsumption by 22% in pH-controlled Bioreactor

• The 19-9 evolved strain utilized 22% of the xylan, consistent with the increases in both rate (15%) and total (16%) H2 production.

• Strain 19-9 displayed a lag in H2 production which can be improved via further adaptation.

• Both the control (∆hpt) and 19-9 mutants were derived from C. thermocellum DSM 1313 which can be manipulated genetically. The benchmark rate (1 L/L-d) was obtained from C. thermocellum ATCC 27405 which lacks a genetic system.

% Utilization 60 100% 50

80%

19-9

hpt

0 12 24 36 48

hpt 19-9

40

H 2 (m

M)

60% 30 20

glucan 40% xylan

10 20% 0

0% Time (hrs)

Results demonstrated bacteria’s capacity to utilize xylan, which makes further improvement promising.

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ApproachTask 3: Generate Metabolic Pathway Mutants

Approach: Redirect metabolic pathways to improve H2 molar yield via developing genetic methods. • ∆pfl: Blocking formate carbon-competing pathway led to 57% increase in rate of

H2 production (2016 AMR accomplishment). • ∆rnf: Manipulating electron-competing pathway led to 35% increase in total H2

production (2017 AMR accomplishment).

Hydrogenase

13C-based metabolic flux analysis could probe metabolic changes responsible for increased H2 production and guide genetic engineering.

Transhydrogenase

Compare growth patterns of the wild-type and mutant strains lacking either the carbon FY19 Q2

Milestone (lactate, formate) competing pathway or deficient in electron inter-conversion (ferredoxin to NADH) by growing them in two different substrates, glucose and cellobiose of different

3/2019

Complete energetics. The outcome will reveal how microbe manage carbon and electron flow toward

increasing H2 production. (NREL). 8

Task 3 Accomplishments and Progress:High-throughput Phenotyping Analysis on H2 Production Mutants

Gen

otyp

e G

row

th P

heno

type

Flux

omic

Phe

noty

pe

H2

∆rnf ∆pfl

1.2

Wei Xiong

• Goal: Unravel metabolic changes Δhyd

1.2 1.2

0.6 0.6 0.6

Δnfn

13C-metabolic flux and growth analysis, 0.4

0.2

0.4 0.4 using high-throughput GC-MS and data 0.2 0.2

0 0 0 automation pipeline 0 20 40 60 0 20 40 60 0 20 40 60

Δhpt 0.8 0.8 0.8

1 1 1 responsible for increased H2 production via

• Progress: (1). Mapped the 13C-flux of Δhpt Δpfl

1.2 1.2 1.2 Δrnf

0.8 0.8 0.8

1 1 1 as the baseline; (2). Analyzed the growth 0.6 0.6 0.6

Δldh

phenotype of H2 production mutants. 0.4 0.4 0.4

0.2 0.2 0.2 The metabolic insights could guide 0 0 0

0 20 40 60 0 20 40 60 0 20 40 60 genetic engineering strategy. Hours NREL | 9

ApproachTask 4: Electrochemically Assisted Microbial Fermentation

Microbial Electrolysis Cell (MEC) — Conversion of Organic Waste to Hydrogen Gas

Goal: Achieving high rate of H2 production with non-Pt based

Effluent from NREL cathode using NREL fermentation reactor fermentation effluent.

(Organic acid rich)

Bruce Logan

Milestones (PSU) Completion Date

Status

FY19

Evaluation of alkaline pH cathode catalysts and select new alkaline-optimized anion exchange membranes. Using a thinner cathode chamber and optimizing hydroxide ions crossover should improve overall performance by 30% (FY18 Q4; PSU*).

*1/2019 – Q4 of Penn State Complete

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Accomplishments and Progress : Alkaline Cathode Catalyst and Membrane Resistance

Goal: determine if alkaline pH could improve H2 production • Use activated carbon Ni (AC-Ni) cathodes with 8.8 mg/cm2 Ni salt loading.

2 g/L acetate H2 Production

pH 7 0.31± 0.02 L/L-d

pH 12 0.4 ± 0.02L/L-d 31% increase

Goal: determine if anion-exchange membrane is the limiting factor • Penn State has developed the “Electrode

Potential Slope (EPS) to quantify internal resistance of various components including membrane.

Resistance mΩ m2

Total 120

Felt Anode 71 ± 5

Solution 25

Cathode 18 ± 2

Membrane 6 ± 5

Conclusion: With its low resistance, membrane is NOT a limiting factor, and will not test/select other membranes per the milestone.

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Accomplishment:Achieve Stable Hydrogen Production of >2 L-H2/L-d Over 90 Day Period

New MEC Reduce number of brush anodes from 7 to 1 to “fill chamber. design:

Anode: 1 brush anode (4.5cm diameter) Cathode: 2 stainless steel wool cathodes Electrolytes: both recirculated

Hydrogen Average (90 d)= 2.62 L-H2/L-d; production Maximum = 3.76 L-H2/L-d

Current Average (138 d) = 197 A/m3

density: Maximum = 414 A/m3

H2 production average rate not yet increased by 30% over previous levels,

but maximum rate was, and reactor stability was greatly improved

Future directions: Increase anode brush diameter to 5.5 cm to completely fill the anode chamber

Emmanuel Fonseca (MS/PhD Student)

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0

0.1

0.2

0.3

0.4

Ni2P/C Ni/C Pt/C

H 2 p

rodu

ctio

n ra

tes

(L/L

/d)

Task 4 Accomplishments and Progress:Cathode Chamber Optimization: Replace Pt with alternative material

Conclusion: Ni2P/C cathode produced 0.29±0.04 L-H2/L-d, comparable to a Pt cathode

Nickel phosphide (Ni2P) nanoparticles on carbon black (CB)

How does Ni2P compare to Pt at pH=7 (vs pH=2) needed for MECs? (Abiotic electrochemical tests.)

Pt/C works better at pH=7 (but also true at pH=2)

(mean ± SD, n=3)

Kyoung-Yeol Kim

Ni2P Nanoparticles Carbon Black

SS mesh

H2

H+

Ni2P/C Cathode

0

2

4

6

8

10

0 10 20 30

Cur

rent

den

sity

(A/m

2 )

Time (h)

Ni2P/C

Ni/C

Pt/C

Avg. Current density

3.5 ± 0.2 A/m2

3.4 ± 0.2 A/m2

3.5 ± 0.3 A/m2

p>0.05, t-test

Result: Insignificant different between

the materials in averaged H2

production (p>0.05)

Result: Initially Ni2P > Ni

(but not as good as Pt)

-16 -14 -12 -10

-8 -6 -4 -2 0

-1.5 -1 -0.5 0

Cur

rent

den

sity

(A/m

2 )

Potential (V vs. Ag/AgCl)

Ni2P/C_pH7 Pt/C_pH7 Ni2P/C_pH2 Pt/C_pH2 pH 2

Max. current of typical MECs

pH 7

MEC tests: How does Ni2P compare to Ni or Pt over a 24

h cycle?

MEC tests: Final results in H2

produced over a 24 h cycle?

(mean±SD,n=3)

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Accomplishments and Progress: Responses to Previous Year Reviewers’ Comments

This project was presented as a poster but was not reviewed during the 2018 AMR

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Collaboration and Coordination

• Task 1 (Bioreactor) Drs. Ali Mohagheghi and Melvin Tucker, National Bioenergy Center at NREL: provide DMR pretreated corn stover and their characterizations - leveraging DOE BETO funding.

• Task 2 (Ionic Liquid) – discontinued in FY17 Drs. Steve Singer (LBNL) and Kent Sales (SNL): conducted biomass pretreatment using ionic liquid as a complementary pretreatment approach to lower feedstock cost.

• Task 3 (Genetic Methods) Dr. James Liao of UCLA in pathway engineering of C. thermocellum – leveraging DOE Office of Science funding.

• Task 4 (MEC) Dr. Bruce Logan at Penn State University: microbial electrolysis cells to improve H2 molar yield.

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Remaining Challenges and Barriers

Task 1. Bioreactor Performance • High solid-substrate loading (175 g/L) is needed to lower H2 selling price, which might

present a challenge to ensure sufficient mixing. This challenge will be addressed by research in a separate project BioHydrogen Consortium,

carried out by Lawrence Berkeley National Lab.

Task 2. Fermentation of Pretreated Biomass using Ionic Liquid (LBNL/SNL) • This task was closed out in FY17/Q1.

Task 3. Generate Metabolic Pathway Mutant in C. thermocellum • Improve the rate of xylan utilization in engineered strain to improve biomass

utilization – addressed by research in a separate project “BioHydrogen Consortium” Continue with adapted evolution strategy feeding xylose/xylan and select fast grower in

xylan. Targeted insertion of foreign genes to overcome the rate-limiting step(s) of xylan utilization.

Task 4. Electrochemically Assisted Microbial Fermentation of Acetate (PSU) • The Penn State subcontract ended in January 17th, 2019.

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Proposed Future Work: project is scheduled toclose out in FY19/Q4

Task 1 (NREL) • Subject 19-9 strain to laboratory adapted evolution by continuously evolving it in avicel

(earlier evolution using cellobiose) and xylan and test H2 production in bioreactors.

Task 2 (LBNL/SNL) • None. Task 2 was discontinued in FY17/Q1. Task 3 (NREL) • Adapt the various C. thermocellum competing-pathway mutants to grow in cellobiose for

comparison of carbon flux channeling through the different metabolic pathways leading to increased H2 production. The outcome will guide metabolic engineering strategies to further improve H2 production (FY19 Q3 Milestone).

Task 4 (Penn State): • None. The Penn State subcontract ended on January 17th, 2019.

Closeout Report. A project closeout report will be submitted to DOE in FY19/Q4, documenting progress in the performance period of FY16-FY19 (FY19 Q4 Milestone).

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Technology Transfer Activities

Technology-to-market or technology transfer plan or strategy • Air Product and Chemicals, Inc.

Main interest in H2 from biomass can be low carbon or even potentially carbon neutral; have funded the Logan lab in the past for work on MECs and RED for H2 production from wastewaters

Large-scale process of greatest interest, but currently there are no larger reactors. Cost needs to be near to, or lower than, making H2 from alternative sources

(natural gas).

Plans for future funding • Pursue opportunities to collaborate with other national Labs and industries

for potential future funding support. • Network with biofuels industry to expand the use of H2. • Advocate the advantages of “green” H2 rather than fossil-fuel derived H2

Patents, licensing • A Record of Invention (ROI-14-70) is filed for developing the proprietary

genetic tools tailored for C. thermocellum. • A second ROI-15-42 has been filed for generating xylose-metabolizing strain,

leading to enhanced biomass utilization.

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Summary

Task 1 • Using a laboratory-evolved strain we observed a 15% increase in rate of H2 production

and 16% in total H2 production by co-fermenting both cellulose and xylan, benchmarking the progress.

• The evolved strain utilized 22% more xylan, which accounts for the above increases. • The outcomes lower the feedstock cost by converting more biomass sugars to H2. Task 2: Closed out in FY17/Q1, not meeting GNG. Task 3 • The genotype of the various mutants were verified via PCR. • Using a 24-well plate Microplate Reader, we performed semi high-throughput growth of

the various C. thermocellum mutants in cellobiose which will be used for 13C-metabolic flux analysis.

Task 4 • Alkaline pH improved H2 production by 31%. • Achieve stable H2 production of >2.6 L-H2/L-d over 90 day period by increasing anode

brush size. • Anion exchange membrane is not a rate-limiting step in H2 production. • Ni2P/C cathode produced 0.29±0.04 L-H2/L-d, comparable to a Pt cathode.

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NREL is a national laboratory of the U.S. Department of Energy, Office of Energy Efficiencyand Renewable Energy, operated by the Alliance for Sustainable Energy, LLC.NREL is a national laboratory of the U.S. Department of Energy, Office of Energy Efficiency

and Renewable Energy, operated by the Alliance for Sustainable Energy, LLC.

Thank You

Publication Number

www.nrel.gov

NREL is a national laboratory of the U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, operated by the Alliance for Sustainable Energy, LLC.

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